Project Summary/Abstract The epidermally-expressed fatty acid binding protein 5 (FABP5) is an intracellular fatty-acid transport protein and a key component of lipid signaling pathways. FABP5 and the epidermal growth factor receptor (EGFR) are both upregulated in a number of cancers including triple negative breast cancer and colorectal cancer. Furthermore, knockdown of FABP5 results in decreased EGFR expression and EGF-induced metastatic potential in triple negative breast cancer cells. In an unbiased proteomics screen for EGF-induced cysteine oxidation, FABP5 cysteine 120 was identified as highly oxidized upon EGF treatment. The results of the screen suggest that FABP5 C120 is reduced in the absence of EGF, but upon EGFR activation, when hydrogen peroxide levels are known to increase within the cell, C120 becomes oxidized. Crystal structures of FABP5 have shown that C120 can form a disulfide bond with C127, proximal to the fatty acid binding pocket of FABP5. The role of disulfide-bond formation on FABP5 function and EGF signaling has not been characterized previously. Here we hypothesize that the EGF-induced formation of the C120-C127 disulfide bond is a mechanism of redox-based EGF signal transduction. Furthermore, we propose that the reactive and redox- active cysteine residues provide a handle for pharmacologically modulating the fatty-acid binding capacity of FABP5 with covalent cysteine-reactive fragments. Therefore, we aim (1) to characterize the influence of disulfide formation on FABP5 fatty acid binding; (2) to measure the impact of FABP5 disulfide formation on EGF signal transduction and cellular cancer phenotypes; and (3) to explore the ligandability of C120/127 on FABP5. In vitro fatty acid binding assays will be performed with oxidized WT FABP5, reduced WT FABP5, as well as a C120S/C127S FABP5 mutant. Cell lines expressing WT FABP5 or the C120S/C127S mutant will be generated and assayed for and EGF signaling and cancer phenotypes. Finally, a mass spectrometry-based screen for covalent ligands of FABP5 C120/C127 will be performed in order to identify specific covalent inhibitors of FABP5 fatty acid binding.